Outbreaks of infectious disease caused by the bacterium Streptococcus pneumoniae, the pneumococcus, were common in the United States early in the 20th century. Today, pneumococcal outbreaks in the U.S. occur in host populations with identifiable risk factors. However, there exist serious concerns that the changing dynamics of pneumococcal populations, brought about by the recent introduction of a conjugate vaccine in the U.S., will broaden the niche of hypervirulent, outbreak-associated strains. The bacterial virulence factors that predispose pneumococci to cause outbreaks are unknown. In this proposal, we will use a molecular epidemiological approach to discover candidate virulence genes in capsular serotype 12F pneumococci that have caused genuine outbreaks in the U.S. and have emerged with hypervirulent characteristics in other countries. We will characterize the clonal diversity of 12F strains using multilocus sequence typing, and we will identify accessory genes in representative strains using suppressive subtractive hybridization (Aim 1). We hypothesize that accessory genes may include virulence genes that contribute to the hypervirulent characteristics of 12F pneumococci. We will screen diverse collections of pneumococci, which were isolated from nasopharyngeal colonization and invasive disease in well-defined host populations, for the discovered accessory genes using multiplex PCR, in order to highlight those genes that are significantly over-represented from invasive disease (Aim 2). This proposal sets up future experimental studies that will address the function of candidate virulence genes that are discovered here. PUBLIC HEALTH RELEVANCE: Capsular serotype 12F pneumococci have caused at least four outbreaks of invasive disease in the United States since 1989. In addition, this serotype has emerged with increased virulence potential in other countries. Revealing the evolutionary origin of these strains and any novel genes linked to their virulence potential will provide basic knowledge about the bacterial factors that promote outbreaks, and may ultimately reveal new mechanisms of bacterial pathogenesis, new markers for surveillance, and new opportunities for disease prevention.